Microfluidic devices have revolutionized analytical separations, facilitating fast analyses with higher resolution, higher efficiency, and lower reagent consumption relative to their macro-scale counterparts [1]. Microchannel-based devices have been used to separate mixtures of analytes ranging from small molecules like amino acids and neurotransmitters to large molecules like DNA and proteins [2]. To complement chemical separations, microchannel-based systems have been developed incorporating pre-column reactions, including enzymatic digestion [3], organic synthesis [4], and fluorescent derivatization [5,6]. These techniques represent the promise of microfluidics for forming fully integrated lab-on-a-chip devices.
Unfortunately, the number and scope of lab-on-a-chip devices capable of integrating pre-column reactions with separations is limited. For example, there are no microfluidic methods reported that are adaptable to shotgun proteomics [7], in which samples are subjected to a rigorous, multi-step processing regimen requiring several days to complete [8]. This deficit is largely mechanistic—managing multiple reagents with precise control over position and reaction time in microchannels is complicated by the near-universal effects of hydrostatic and capillary flows [9-11]. The development of integrated microvalves [12] offers some relief from this problem; however, the complicated fabrication and control infrastructure required for this technology has limited its widespread use [13]. Another technique that might be useful for pre-column reactions and separations is multi-phase microfluidic systems (i.e., droplets in channels) [14]. In recent work, Edgar et al. [15] and Roman et al. [16] reported methods capable of delivering droplets from such systems directly into separation channels. This is an exciting new development, but the droplets-in-channels paradigm is not ideally suited for controlling chemical reactions, as droplets (regardless of their contents) are controlled in series.
An alternative miniaturized fluid handling format to microchannels is digital microfluidics (DMF), a technique in which discrete fluidic droplets are manipulated by electrostatic forces on an array of electrodes coated with an insulating dielectric [17-19]. DMF is well-suited for carrying out sequential chemical reactions in which droplets containing different reagents [20,21] and phases [22] can be dispensed from reservoirs, moved, merged, mixed and split [23]. For example, it was recently shown that a multistep proteomic sample processing workup can be achieved by digital microfluidics, in which protein samples were sequentially reduced, alkylated, and digested [24]. Likewise, a DMF method to purify proteins from serum in a multistep process comprising precipitation, rinsing, and resolubilization has also been implemented [25]. These types of sequential processing regimens are difficult to implement using microchannels.
Unfortunately, prior art adaptations of digital microfluidic technology to separation and other analytical methods have focused on the use of capillaries. Capillaries are broadly known in the art as being distinct from microchannel devices. Specifically, capillaries are tubular structures having an inner and outer diameter in which the inner diameter is sufficiently small to promote the flow of liquid by capillary action.
One example of a DMF-capillary device is postulated by Fair [26], wherein there is a description of a platform involving the transfer of sample from an electrowetting-on-dielectric device to a capillary electrophoresis device that includes a capillary for separation. This scheme includes two separate devices, namely a DMF array for sample pre-processing and a capillary device for separation. The use of two separate devices leads to a host of technical difficulties not addressed in [26], including the precise spatial alignment required to achieve flow from one device to another, and difficulty in adapting and securing capillaries for use with a DMF elements.
Another capillary-DMF device is provided in International Patent Application WO/2009/111431, which provides a DMF array that is connected to a capillary for transferring liquid to another physically separate analysis device. In specific embodiments, the DMF device is adapted to an electrospray ionization compatible tip to allow interfacing with a mass spectrometer. Unfortunately, such a device presents numerous practical challenges including the integration of a tubular capillary with a planar DMF device. Further, the spatial extension of the capillary beyond the planar DMF substrate presents a very high risk of breakage. This high risk of breakage is further exacerbated by designs in which a capillary is suspended below the DMF substrate, which would almost certainly lead to breakage during routine use.
An improved device adapting electrowetting technology to microchannels is provided by International Patent Application No. WO 2007/048111, which discloses a microfluidic channel that incorporates electrowetting for the extraction of separated species. The device includes a microfluidic channel, with electrodes located at either end of the channel that draw in sample for electrophoretic separation. In addition, the microfluidic channel includes a wall opening located along the channel, where electrowetting is employed to extract a target separated along the channel. The wall opening can be provided anywhere along a non-walled liquid column or at any wall opening in between the ends of a wall-bounded liquid channel. While extracting a droplet from one side of the channel, a second refill droplet is added on the other side of the channel to prevent remixing of separated species.
Although this device succeeds in providing droplet-based extraction, it suffers from a number of disadvantages that limit its practical use as a microfluidic separation device. First, extracting droplets from a channel used for a chemical separation will destroy the resolution gained in the separation as the separated components will recombine as the droplets are sampled off the channel. Second, and most notably, all initial sample processing must be performed off-line, using either manual methods or another automated system.
Accordingly, it would therefore be advantageous to provide an improved microfluidic device that provides the capability of both sample pre-processing and microfluidic separation without suffering from the problems associated with aligning multiple devices and without requiring the use of tubular capillaries for separation.